GGT5: a potential immunotherapy response inhibitor in gastric cancer by modulating GSH metabolism and sustaining memory CD8+ T cell infiltration.

PURPOSE: The variable responses to immunotherapy observed in gastric cancer (GC) patients can be attributed to the intricate nature of the tumor microenvironment. Glutathione (GSH) metabolism significantly influences the initiation and progression of gastric cancer. Consequently, targeting GSH metabolism holds promise for improving the effectiveness of Immune checkpoints inhibitors (ICIs). METHODS: We investigated 16 genes related to GSH metabolism, sourced from the MSigDB database, using pan-cancer datasets from TCGA. The most representative prognosis-related gene was identified for further analysis. ScRNA-sequencing analysis was used to explore the tumor heterogeneity of GC, and the results were confirmed by  Multiplex immunohistochemistry (mIHC). RESULTS: Through DEGs, LASSO, univariate and multivariate Cox regression analyses, and survival analysis, we identified GGT5 as the hub gene in GSH metabolism with the potential to promote GC. Combining CIBERSORT, ssGSEA, and scRNA analysis, we constructed the immune architecture of GC. The subpopulations of T cells were isolated, revealing a strong association between GGT5 and memory CD8+ T cells. Furthermore, specimens from 10 GC patients receiving immunotherapy were collected. mIHC was used to assess the expression levels of GGT5 and memory CD8+ T cell markers. Our results established a positive correlation between GGT5 expression, the enrichment of memory CD8+ T cells, and a suboptimal response to immunotherapy. CONCLUSIONS: Our study identifies GGT5, a hub gene in GSH metabolism, as a potential therapeutic target for inhibiting the response to immunotherapy in GC patients. These findings offer new insights into strategies for optimizing immunotherapy of GC.

Rotini-like MXene@LCE Actuator with Diverse and Programmable Actuation Based on Dual-mode Synergy.

Liquid crystalline elastomer (LCE) exhibits muscle-like actuation upon order-disturbed stimulus, offering ample room for designing soft robotic systems. Multimodal LCE is demonstrated to unleash the potential to perform multitasks. However, each actuation mode is typically isolated. In contrast, coordination between different actuation modes based on an MXene-doped LCE is realized, whose actuation can be triggered either by directly heating/cooling or using near-infrared light due to the photo-thermal effect of MXene. As such, the two activation modes (heat and light) not only can work individually to offer stable actuation under different conditions but also can collaborate synergistically to generate more intelligent motions, such as achieving the brake and turn of an autonomous rolling. The principle therefore can diversify the design principles for multifunctional soft actuators and robotics.

Porous Organic Framework Materials (MOF, COF, and HOF) as the Multifunctional Separator for Rechargeable Lithium Metal Batteries.

The separator is an important component in batteries, with the primary function of separating the positive and negative electrodes and allowing the free passage of ions. Porous organic framework materials have a stable connection structure, large specific surface area, and ordered pores, which are natural places to store electrolytes. And these materials with specific functions can be designed according to the needs of researchers. The performance of porous organic framework-based separators used in rechargeable lithium metal batteries is much better than that of polyethylene/propylene separators. In this paper, the three most classic organic framework materials (MOF, COF, and HOF) are analyzed and summarized. The applications of MOF, COF, and HOF separators in lithium-sulfur batteries, lithium metal anode, and solid electrolytes are reviewed. Meanwhile, the research progress of these three materials in different fields is discussed based on time. Finally, in the conclusion, the problems encountered by MOF, COF, and HOF in different fields as well as their future research priorities are presented. This review will provide theoretical guidance for the design of porous framework materials with specific functions and further stimulate researchers to conduct research on porous framework materials.

Hydrogen-Bonded Organic Framework as Superior Separator with High Lithium Affinity C═N Bond for Low N/P Ratio Lithium Metal Batteries.

Lithium dendrite and side reactions are two major challenges for lithium metal anode. Here, the highly lithophilic triazine ring in the hydrogen-bonded organic framework is recommended to accelerate the desolvation process of lithium ions. Among them, the formation of Li-N bonds between lithium ions and the triazine ring in CAM reduces the diffusion energy barrier of Li+ crossing the SEI interface and the desolvation energy barrier of Li+ exiting from the solvent sheath so that the rapid and homogeneous deposition of lithium-ion can be achieved. Meanwhile, the lithium-ion migration coefficient can be as high as 0.70. CAM separator is used to assemble lithium metal batteries with nickel-rich cathodes (NCM 622). When N/P = 8 and 5, the capacity retention rates of Li-NCM 622 full cell are 78.2% and 80.5% after 200 and 110 cycles, respectively, and the Coulomb efficiency can be maintained at 99.5%, showing excellent cycle stability.

[Progress in antibacterial coatings of titanium implants surfaces].

In recent years, bone implant materials such as titanium and titanium alloys have been widely used in the biomedical field due to their excellent mechanical properties and good biocompatibility. However, in clinical practice, bacterial adhesion to the material surface and postoperative infection issues may lead to implantation failure. Based on the antibacterial mechanism, this review elaborated on the antibacterial surface design of titanium implants from the aspects of anti-bacterial adhesion, contact sterilization and photocontrol sterilization. Surface modification of titanium or titanium-based alloy implants with different techniques can inhibit bacteria and promote osseointegration. Thus, the application range of multifunctional titanium-based implants in the field of orthopedics will be expanded.

Spontaneous Built-In Electric Field in C3 N4 -CoSe2 Modified Multifunctional Separator with Accelerating Sulfur Evolution Kinetics and Li Deposition for Lithium-Sulfur Batteries.

The discovery of the heterostructures that is combining two materials with different properties has brought new opportunities for the development of lithium sulfur batteries (LSBs). Here, C3 N4 -CoSe2 composite is elaborately designed and used as a functional coating on the LSBs separator. The abundant chemisorption sites of C3 N4 -CoSe2 form chemical bonding with polysulfides, provides suitable adsorption energy for lithium polysulfides (LiPSs). More importantly, the spontaneously formed internal electric field accelerates the charge flow in the C3 N4 -CoSe2 interface, thus facilitating the transport of LiPSs and electrons and promoting the bidirectional conversion of sulfur. Meanwhile, the lithiophilic C3 N4 -CoSe2 sample with catalytic activity can effectively regulate the uniform distribution of lithium when Li+ penetrates the separator, avoiding the formation of lithium dendrites in the lithium (Li) metal anode. Therefore, LSBs based on C3 N4 -CoSe2 functionalized membranes exhibit a stable long cycle life at 1C (with capacity decay of 0.0819% per cycle) and a large areal capacity of 10.30 mAh cm-2 at 0.1C (sulfur load: 8.26 mg cm-2 , lean electrolyte 5.4 µL mgs -1 ). Even under high-temperature conditions of 60 °C, a capacity retention rate of 81.8% after 100 cycles at 1 C current density is maintained.

MIR-107/HMGB1/FGF-2 axis responds to excessive mechanical stretch to promote rapid repair of vascular endothelial cells.

The increase of vascular wall tension can lead to endothelial injury during hypertension, but its potential mechanism remains to be studied. Our results of previous study showed that HUVECs could induce changes in HMGB1/RAGE to resist abnormal mechanical environments in pathological mechanical stretching. In this study, we applied two different kinds of mechanical tension to endothelial cells using the in vitro mechanical loading system FlexCell-5000T and focused on exploring the expression of miR-107 related pathways in HUVECs with excessive mechanical tension. The results showed that miR-107 negatively regulated the expression of the HMGB1/RAGE axis under excessive mechanical tension. Excessive mechanical stretching reduced the expression of miR-107 in HUVECs, and increased the expression of the HMGB1/RAGE axis. When miR-107 analog was transfected into HUVECs with lipo3000 reagent, the overexpression of miR-107 slowed down the increase of the HMGB1/RAGE axis caused by excessive mechanical stretching. At the same time, the overexpression of miR-107 inhibited the proliferation and migration of HUVECs to a certain extent. On the contrary, when miR-107 was silent, the proliferation and migration of HUVECs showed an upward trend. In addition, the study also showed that under excessive mechanical tension, miR-107 could regulate the expression of FGF-2 by HMGB1. In conclusion, these findings suggest that pathological mechanical stretching promote resistance to abnormal mechanical stimulation on HUVECs through miR-107/HMGB1/RAGE/FGF-2 pathway, thus promote vascular repair after endothelial injury. The suggest that miR-107 is a potential therapeutic target for hypertension.

[Progress in preparation and application of sodium alginate microspheres].

Sodium alginate (SA) is a kind of natural polymer material extracted from kelp, which has excellent biocompatibility, non-toxicity, biodegradability and abundant storage capacity. The formation condition of sodium alginate gel is mild, effectively avoiding the inactivation of active substances. After a variety of preparation methods, sodium alginate microspheres are widely used in the fields of biomaterials and tissue engineering. This paper reviewed the common methods of preparing alginate microspheres, including extrusion, emulsification, electrostatic spraying, spray drying and coaxial airflow, and discussed their applications in biomedical fields such as bone repair, hemostasis and drug delivery.

Feedforward regulation of Myc coordinates lineage-specific with housekeeping gene expression during B cell progenitor cell differentiation.

The differentiation of self-renewing progenitor cells requires not only the regulation of lineage- and developmental stage-specific genes but also the coordinated adaptation of housekeeping functions from a metabolically active, proliferative state toward quiescence. How metabolic and cell-cycle states are coordinated with the regulation of cell type-specific genes is an important question, because dissociation between differentiation, cell cycle, and metabolic states is a hallmark of cancer. Here, we use a model system to systematically identify key transcriptional regulators of Ikaros-dependent B cell-progenitor differentiation. We find that the coordinated regulation of housekeeping functions and tissue-specific gene expression requires a feedforward circuit whereby Ikaros down-regulates the expression of Myc. Our findings show how coordination between differentiation and housekeeping states can be achieved by interconnected regulators. Similar principles likely coordinate differentiation and housekeeping functions during progenitor cell differentiation in other cell lineages.

Role of sphingosine-1-phosphate receptors in vascular injury of inflammatory bowel disease.

Sphingosine-1-phosphate receptors (S1PRs) have an impact on the intestinal inflammation of inflammatory bowel disease (IBD) by regulating lymphocyte migration and differentiation. S1PR modulators as an emerging therapeutic approach are being investigated for the treatment of IBD. However, the role of S1PRs in intestinal vessels has not drawn much attention. Intestinal vascular damage is one of the major pathophysiological features of IBD, characterized by increased vascular density and impaired barrier function. S1PRs have pleiotropic effects on vascular endothelial cells, including proliferation, migration, angiogenesis and barrier homeostasis. Mounting evidence shows that S1PRs are abnormally expressed on intestinal vascular endothelial cells in IBD. Unexpectedly, S1PR modulators may damage intestinal vasculature, for example increase intestinal bleeding; therefore, S1PRs are thought to be involved in the regulation of intestinal vascular function in IBD. However, little is understood about how S1PRs regulate intestinal vascular function and participate in the initiation and progression of IBD. In this review, we summarize the pathogenic role of S1PRs in and the underlying mechanisms behind the intestinal vascular injury in IBD in order for improving IBD practice including S1PR-targeted therapies.

IL-17A secreted from lymphatic endothelial cells promotes tumorigenesis by upregulation of PD-L1 in hepatoma stem cells.

BACKGROUND & AIMS: The microenvironment regulates hepatoma stem cell behavior. However, the contributions of lymphatic endothelial cells to the hepatoma stem cell niche remain largely unknown; we aimed to analyze this contribution and elucidate the mechanisms behind it. METHODS: Associations between lymphatic endothelial cells and CD133+ hepatoma stem cells were analyzed by immunofluorescence and adhesion assays; with the effects of their association on IL-17A expression examined using western blot, quantitative reverse transcription PCR and luciferase reporter assay. The effects of IL-17A on the self-renewal and tumorigenesis of hepatoma stem cells were examined using sphere and tumor formation assays. The role of IL-17A in immune escape by hepatoma stem cells was examined using flow cytometry. The expression of IL-17A in hepatoma tissues was examined using immunohistochemistry. RESULTS: CD133+ hepatoma stem cells preferentially interact with lymphatic endothelial cells. The interaction between the mannose receptor and high-mannose type N-glycans mediates the interaction between CD133+ hepatoma stem cells and lymphatic endothelial cells. This interaction activates cytokine IL-17A expression in lymphatic endothelial cells. IL-17A promotes the self-renewal of hepatoma stem cells. It also promotes their immune escape, partly through upregulation of PD-L1. CONCLUSION: Interactions between lymphatic endothelial cells and hepatoma stem cells promote the self-renewal and immune escape of hepatoma stem cells, by activating IL-17A signaling. Thus, inhibiting IL-17A signaling may be a promising approach for hepatoma treatment. LAY SUMMARY: The microenvironment is crucial for the self-renewal and development of hepatoma stem cells, which lead to the development of liver cancer. Lymphatic endothelial cells are an important component of this niche microenvironment, helping hepatoma stem cells to self-renew and escape immune attack, by upregulating IL-17A signaling. Thus, targeting IL-17A signaling is a potential strategy for the treatment of hepatoma.

Genome-wide identification and analysis of the EIN3/EIL gene family in allotetraploid Brassica napus reveal its potential advantages during polyploidization.

BACKGROUND: Polyploidization is a common event in the evolutionary history of angiosperms, and there will be some changes in the genomes of plants other than a simple genomic doubling after polyploidization. Allotetraploid Brassica napus and its diploid progenitors (B. rapa and B. oleracea) are a good group for studying the problems associated with polyploidization. On the other hand, the EIN3/EIL gene family is an important gene family in plants, all members of which are key genes in the ethylene signaling pathway. Until now, the EIN3/EIL gene family in B. napus and its diploid progenitors have been largely unknown, so it is necessary to comprehensively identify and analyze this gene family. RESULTS: In this study, 13, 7 and 7 EIN3/EIL genes were identified in B. napus (2n = 4x = 38, AnCn), B. rapa (2n = 2x = 20, Ar) and B. oleracea (2n = 2x = 18, Co). All of the identified EIN3/EIL proteins were divided into 3 clades and further divided into 8 sub-clades. Ka/Ks analysis showed that all identified EIN3/EIL genes underwent purifying selection after the duplication events. Moreover, gene structure analysis showed that some EIN3/EIL genes in B. napus acquired introns during polyploidization, and homolog expression bias analysis showed that B. napus was biased towards its diploid progenitor B. rapa. The promoters of the EIN3/EIL genes in B. napus contained more cis-acting elements, which were mainly involved in endosperm gene expression and light responsiveness, than its diploid progenitors. Thus, B. napus might have potential advantages in some biological aspects. CONCLUSIONS: The results indicated allotetraploid B. napus might have potential advantages in some biological aspects. Moreover, our results can increase the understanding of the evolution of the EIN3/EIL gene family in B. napus, and provided more reference for future research about polyploidization.

SAE1 promotes human glioma progression through activating AKT SUMOylation-mediated signaling pathways.

BACKGROUND: The SUMO-activating enzyme SAE1 is indispensable for protein SUMOylation. A dysregulation of SAE1 expression involves in progression of several human cancers. However, its biological roles of SAE1 in glioma are unclear by now. METHODS: The differential proteome between human glioma tissues and para-cancerous brain tissues were identified by LC-MS/MS. SAE1 expression was further assessed by immunohistochemistry. The patient overall survival versus SAE1 expression level was evaluated by Kaplan-Meier method. The glioma cell growth and migration were evaluated under SAE1 overexpression or inhibition by the CCK8, transwell assay and wound healing analysis. The SUMO1 modified target proteins were enriched from total cellular or tissue proteins by incubation with the anti-SUMO1 antibody on protein-A beads overnight, then the SUMOylated proteins were detected by Western blot. Cell apoptosis and cell cycle were analyzed by flow cytometry. The nude mouse xenograft was determined glioma growth and tumorigenicity in vivo. RESULTS: SAE1 is identified to increase in glioma tissues by a quantitative proteomic dissection, and SAE1 upregulation indicates a high level of tumor malignancy grade and a poor overall survival for glioma patients. SAE1 overexpression induces an increase of the SUMOylation and Ser473 phosphorylation of AKT, which promotes glioma cell growth in vitro and in nude mouse tumor model. On the contrary, SAE1 silence induces an obvious suppression of the SUMOylation and Ser473 phosphorylation of Akt, which inhibits glioma cell proliferation and the tumor xenograft growth through inducing cell cycle arrest at G2 phase and cell apoptosis driven by serial biochemical molecular events. CONCLUSION: SAE1 promotes glioma cancer progression via enhancing Akt SUMOylation-mediated signaling pathway, which indicates targeting SUMOylation is a promising therapeutic strategy for human glioma.

H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling in HEK293 cells.

Mammalian target of rapamycin (mTOR) is a Ser/Thr protein kinase that functions as an ATP and amino acid sensor to govern cell growth and proliferation by mediating mitogen- and nutrient-dependent signal transduction. Protein phosphatase 2A (PP2A), a ubiquitously expressed serine/threonine phosphatase, negatively regulates mTOR signaling. Methylation of PP2A is catalyzed by leucine carboxyl methyltransferase-1 (LCMT1) and reversed by protein phosphatase methylesterase 1 (PME-1), which regulates PP2A activity and substrate specificity. However, whether PP2A methylation is related to mTOR signaling is still unknown. In this study, we examined the effect of PP2A methylation on mTOR signaling in HEK293 cells under oxidative stress. Our results show that oxidative stress induces PP2A demethylation and inhibits the mTORC1 signaling pathway. Next, we examined two strategies to block PP2A demethylation under oxidative stress. One strategy was to prevent PP2A demethylation using a PME-1 inhibitor; the other strategy was to activate PP2A methylation via overexpression of LCMT1. The results show that both the PME-1 inhibitor and LCMT1 overexpression prevent the mTORC1 signaling suppression induced by oxidative stress. Additionally, LCMT1 overexpression rescued cell viability and the mitochondrial membrane potential decrease in response to oxidative stress. These results demonstrate that H2 O2 induces PP2A demethylation to downregulate mTORC1 signaling. These findings provide a novel mechanism for the regulation of PP2A demethylation and mTORC1 signaling under oxidative stress.

Characterization of overexpression of the alternatively spliced isoform of the protein phosphatase 2A catalytic subunit in cells.

PP2Acα2 is a recently discovered PP2Acα alternative splicing isoform that can be induced following serum withdrawal. It shows enhanced binding to immunoglobulin binding protein 1 and is overexpressed in chronic lymphocytic leukemia patients. Current knowledge concerning PP2Acα2 is limited. In this study, we induced and cloned PP2Acα2 from HL-60 cells and human lymphocytes, transfected them into human embryonic kidney 293 cells and constructed a stable overexpression cell line. We found that PP2Acα2 mRNA inhibits expression of its longer isoform PP2Acα mRNA but had no effect on the final protein expression and modification of this longer isoform. Moreover, PP2Acα2-overexpressed cells demonstrated increased expression of IGBP1, activated mTORC1 signaling to reduce basal autophagy and increased anchorage-independent growth. Our study provides new insights into the complex mechanisms of PP2A regulation.

Downregulation of ARHGDIA contributes to human glioma progression through activation of Rho GTPase signaling pathway.

The protein ARHGDIA has been found to play distinct roles in cancer progression for several tumors. However, it remains elusive whether and how ARHGDIA plays functions in human glioma. In this study, we discovered that ARHGDIA is much downregulated in human glioma; meanwhile, its expression negatively correlates with glioma malignancy and positively relates to prognosis of glioma patients. It has independent predictive value of ARHGDIA expression level for overall survival of human glioma patients. Glioma patients with ARHGDIA-positive expression have a longer overall survival time than ARHGDIA-negative patients. Knockdown of ARHGDIA promotes cell proliferation, cell cycle progression, and cell migration due to the activation of Rho GTPases (Rac1, Cdc42, and RhoA) and Akt phosphorylation, whereas overexpression of ARHGDIA suppresses cell growth, cell cycle progression, and cell migration. ARHGDIA is a potential prognostic marker and therapeutic target for human glioma.

Genome-wide identification of Ikaros targets elucidates its contribution to mouse B-cell lineage specification and pre-B-cell differentiation.

Ikaros family DNA-binding proteins are critical regulators of B-cell development. Because the current knowledge of Ikaros targets in B-cell progenitors is limited, we have identified genes that are bound and regulated by Ikaros in pre-B cells. To elucidate the role of Ikaros in B-cell lineage specification and differentiation, we analyzed the differential expression of Ikaros targets during the progression of multipotent to lymphoid-restricted progenitors, B- and T-cell lineage specification, and progression along the B-cell lineage. Ikaros targets accounted for one-half of all genes up-regulated during B-cell lineage specification in vivo, explaining the essential role of Ikaros in this process. Expression of the Ikaros paralogs Ikzf1 and Ikzf3 increases incrementally during B-cell progenitor differentiation, and, remarkably, inducible Ikaros expression in cycling pre-B cells was sufficient to drive transcriptional changes resembling the differentiation of cycling to resting pre-Bcells in vivo. The data suggest that Ikaros transcription factor dosage drives the progression of progenitors along a predetermined lineage by regulating multiple targets in key pathways, including pre-B­cell receptor signaling, cell cycle progression, and lymphocyte receptor rearrangement.Our approachmay be of general use to map the contribution of transcription factors to cell lineage commitment and differentiation.

Association of preoperative elevated lipoprotein (a) with poor survival in patients with biliary tract cancers.

BACKGROUND: Biliary tract cancers have garnered significant attention due to their highly malignant nature. The relationship between abnormal lipid metabolism and tumor occurrence and development is a research hotspot. However, its correlation with biliary tract cancers is unclear. METHODS: We enrolled 78 patients with biliary tract cancers and obtained data on clinical characteristics, pathological findings, and preoperative blood lipid indices, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), triglycerides (TG), and lipoprotein (a) [Lp(a)]. Receiver operating characteristic (ROC) curves were used to determine the optimal predictive cutoff values of lipid indicators among the participants. Independent risk factors were determined using Cox regression, and survival was predicted using the Kaplan-Meier method. Statistical analyses were performed using SPSS software. RESULTS: Univariate Cox regression analysis revealed that the body mass index (BMI), tumor location, surgical margin, N stage, and abnormally increased LDL-C, TG, and Lp(a) levels were significantly associated with poor prognosis of biliary tract cancers (p < 0.05). Multifactor Cox regression demonstrated that only N stage (HR = 3.393, p < 0.001) and abnormally increased Lp(a) levels (HR = 2.814, p = 0.004) were significantly associated with shorter survival. N stage and Lp(a) were identified as independent prognostic risk factors for patients with biliary tract cancers. CONCLUSION: This study presents Lp(a) as a novel biochemical marker that can guide clinical treatment strategies for patients with biliary tract cancers. More effective treatment options and intensive postoperative testing should be considered to prolong the survival of these patients with preoperative abnormal lipid metabolism.

In vitro corrosion and cytocompatibility of Mg-Zn-Ca alloys coated with FHA.

In the field of orthopedics, it's crucial to effectively slow down the degradation rate of Mg alloys. This study aims to improve the degradation behavior of Mg-Zn-Ca alloys by electrodepositing fluorohydroxyapatite (FHA). We investigated the microstructure and bond strength of the deposition, as well as degradation and cellular reactions. After 15-30 days of degradation in Hanks solution, FHA deposited alloys showed enhanced stability and less pH change. The strong interfacial bond between FHA and the Mg-Zn-Ca substrate was verified through scratch tests (Critical loads: 10.73 ± 0.014 N in Mg-Zn-0.5Ca alloys). Cellular studies demonstrated that FHA-coated alloys exhibited good cytocompatibility and promoted the growth of MC3T3-E1 cells. Further tests showed FHA-coated alloys owed improved early bone mineralization and osteogenic properties, especially in Mg-Zn-0.5Ca. This research highlighted the potential of FHA-coated Mg-Zn-0.5Ca alloys in orthopedics applications.

Targeting the PI3K/AKT/mTOR pathway offer a promising therapeutic strategy for cholangiocarcinoma patients with high doublecortin-like kinase 1 expression.

BACKGROUND: Cholangiocarcinoma (CCA), characterized by high heterogeneity and extreme malignancy, has a poor prognosis. Doublecortin-like kinase 1 (DCLK1) promotes a variety of malignant cancers in their progression. Targeting DCLK1 or its associated regulatory pathways can prevent the generation and deterioration of several malignancies. However, the role of DCLK1 in CCA progression and its molecular mechanisms remain unknown. Therefore, we aimed to investigate whether and how DCLK1 contributes to CCA progression. METHODS: The expression of DCLK1 in CCA patients was detected using Immunohistochemistry (IHC). We established DCLK1 knockout and DCLK1 overexpression cell lines for Colony Formation Assay and Transwell experiments to explore the tumor-promoting role of DCLK1. RT-PCR, Western blot and multiple fluorescent staining were used to assess the association between DCLK1 and epithelial-mesenchymal transition (EMT) markers. RNA sequencing and bioinformatics analysis were performed to identify the underlying mechanisms by which DCLK1 regulates CCA progression and the EMT program. RESULTS: DCLK1 was overexpressed in CCA tissues and was associated with poor prognosis. DCLK1 overexpression facilitated CCA cell invasion, migration, and proliferation, whereas DCLK1 knockdown reversed the malignant tendencies of CCA cells, which had been confirmed both in vivo and in vitro. Furthermore, we demonstrated that DCLK1 was substantially linked to the advancement of the EMT program, which included the overexpression of mesenchymal markers and the downregulation of epithelial markers. For the underlying mechanism, we proposed that the PI3K/AKT/mTOR pathway is the key process for the role of DCLK1 in tumor progression and the occurrence of the EMT program. When administered with LY294002, an inhibitor of the PI3K/AKT/mTOR pathway, the tumor's ability to proliferate, migrate, and invade was greatly suppressed, and the EMT process was generally reversed. CONCLUSIONS: DCLK1 facilitates the malignant biological behavior of CCA cells through the PI3K/AKT/mTOR pathway. In individuals with cholangiocarcinoma who express DCLK1 at high levels, inhibitors of the PI3K/AKT/mTOR signaling pathway may be an effective therapeutic approach.